Near-field optical probes are valuable tools for the characterization of complex structures and processes in solid state materials, soft matter and biological samples that occur over length scales smaller than the wavelength of light. In variants of near-field scanning optical microscopy (NSOM), an optical probe is typically integrated with a scanning tip and rastered over a sample to form an image. Images acquired with NSOM typically require mechanical scanning and can contain artifacts from tip-sample interactions. Alternatively, in scanning electron microscopy (SEM), a focused electron beam is electronically scanned over a sample to obtain nanoscale images by correlating the detected scattered electrons with the position of the beam. Traditional electron microscopy does not use spectrally-specific excitation and can damage soft materials, such as biological samples.
Alternatively, cathodoluminescence (CL) may also be used for nanoscale imaging. Current CL methods typically detect the light generated in the sample by the application of an electron beam. CL has been used to investigate the nanoscale properties of solid luminescent materials and to characterize metallic nanostructures. Direct CL has also been used to image biological samples, but typically causes damage to the sample and can result in poor imaging quality. Methods which incorporate inorganic cathodoluminescent nanoparticle labels into a biological sample result in less sample damage, but imaging with nanoparticle labels requires the electron beam to penetrate into the sample, which precludes repeated measurements or observations of dynamics. Further, the structure of some samples, including biological and nonbiological samples, makes it difficult to attach fluorescent labels, limiting their ability to be imaged.
Thus, various methods are known in the art to image samples; however, capturing nanoscale sample dynamics is a challenge in the art. What is needed in the art are alternative methods that can achieve speed and is suitable for prolonged imaging. For example, what is needed is a method of nanoscale imaging in which electron beam damage to the sample is minimized, the sample can be imaged quickly, and the sample can be imaged multiple times.